1. Field of the Invention
The present invention relates to an apparatus containing microarray binding sensors having biological probe materials using carbon nanotube transistors and various methods for detecting binding of biological target materials thereto.
2. Description of the Background
DNA microarrays are powerful tools in molecular biology, and generally contain an array of hundreds to tens of thousands of genes spotted on a solid substrate, and which is used to identify and quantify unknown gene samples. The microarray technique is predicated upon the property that nucleic acid hybridization is highly specific, i.e., cytosine binds only to guanine and thymine to adenine. Thus, a specific sequence of nucleic acids, for example, 5′ ATCATC3,′ will preferentially bind with its complementary sequence, 3′ TAGTAG5.′
DNA microarrays are invaluable techniques for high throughput monitoring of gene expression at the transcription level, determining genome wide DNA copy number changes, identifying targets of transcription factors, sequencing and, more recently, for profiling micro RNA (miRNA) levels in cancer. The central dogma in molecular biology is that DNA is transcribed to ribonucleic acid (RNA), and the information in the RNA is used to make proteins, by a process called translation. Since the function and metabolism of the cell is regulated by the protein produced in the cell, many diseases caused by gene mutations, such as cancers, can be studied by monitoring the gene expression. Thus, the identification and quantification of genes is of particular interest. It is important to know the particular gene or genes that contribute to a certain phenotype, and also the amount of the gene that signifies the level of the gene expression. There are diseases, however, which are not necessarily caused by gene mutation or change in DNA sequence, but which are caused by an abnormal amount of the gene or abnormal level of gene expression. High throughput gene identification enables researchers to quickly identify the genes that undergo mutations in a certain disease. Comparative gene expression compares the level of gene expression, between a cancerous cell and a healthy cell, for example. In a typical DNA microarray experiment that relies on fluorescent detection, comparative gene expression is done by labeling the genes in one cell with one color of fluorescent reporter molecules, and genes in the other cell with another. The relative intensity of each color is a direct measure of the abundance of the genes from the two cells. Given the versatility of DNA microarrays, the impact thereof on healthcare is expected to be quite significant if DNA microarrays can be deployed widely and inexpensively. It will enable rapid diagnosis of diseases, as well as enable drugs to be tailored to each patient to achieve highest effectiveness.
The first reported DNA microarray was fabricated on nylon membranes using cDNA clones and utilized radioactively labeled targets for detection. Since then, many large-scale DNA microarray platforms have been developed, which have included, double-stranded cDNA, single stranded short 25mers (Affymetrix), mid-sized 30mer (Combimatrix) or long 50-70mers (Nimblegen or Agilent) oligonucleotides. All of these methods rely upon various combinations of enzymatic amplification of the nucleic acid and fluorescent labeling of targets, hybridization, and amplification of signal followed by detection by optical scanners.
In a microarray experiment, an array of known single stranded DNA sequences, called probes, is immobilized on a substrate and later exposed to an unknown set of target genes (or single stranded DNA sequences) that have been chemically tagged with fluorescent molecules. In places on the array where the probe and target sequences are complementary, hybridization occurs and the locations of these specific binding events are reported by the fluorescent molecules.
A major hurdle of using DNA microarray as a clinical tool is that the technique is laborious, requires complex protocols, requires large amounts of reagents, and suffers from low signal to noise ratio and rapid optical degradation. While significant strides have been made in fluorescent-based DNA microarray technology, the methodologies are often time-consuming and in addition rely on the determination of fluorescence intensity and the sensitivity is thus limited by the ability to detect small numbers of photons. Moreover, fluorescent molecules suffer from photobleaching, which means that the fluorescent molecule will stop to fluoresce after receiving a certain amount of excitation.
A variety of DNA detection schemes has been reported in the literature. The detection mechanisms involve detection of the existence of the reporter molecules or tags, such as radioisotopes, fluorophores, quantum dots, gold nanoparticles, magnetic nanoparticles, or enzymes, for example. A brief survey of known fluorescent based DNA microarray, and other label-free electronic field effect DNA detection schemes is described below in subsections 1) and 2).
1. Fluorescence-Based Microarrays
Typically, microarrays are microscope glass slides spotted with thousands of different genes. The array does not have built-in reader. The detection is performed using a fluorescence scanner after hybridization with fluorescent-tagged target DNA. There are two ways to make microarrays: (i) spotting cDNA or oligonucleotides onto the substrate with a robotic spotter, or (ii) direct oligonucleotide synthesis on the solid support. A robotic spotter uses thousands of capillary pins dipped into wells containing different kind of genes and transports the genes onto a functionalized solid substrate to create gene spots. Another approach, such as the one employed by affymetrix, uses direct oligonucleotide synthesis on the substrate. The ingredients are solutions of the four nucleotides: adenine, guanine, cytosine and thymine which bear light sensitive protecting group. The process starts with a quartz wafer that is coated with a light-sensitive chemical compound that prevents coupling between the wafer and the first nucleotide of the DNA probe being created. Lithographic masks are used to either block or transmit light onto specific locations of the wafer surface. The exposed spots are now ready to couple with a nucleotide. The surface is then flooded with a solution containing either adenine, thymine, cytosine, or guanine, and coupling occurs only in those regions on the glass that have been deprotected through illumination. The coupled nucleotide also bears a light-sensitive protecting group, so the cycle of deprotection and coupling until the probes reach their full length, usually 25 nucleotides.
2. Field Effect DNA Detection
In general, many field effect based biomolecule detection schemes resemble the structure of ISFET (ion sensitive field effect transistor), which was first introduced by Bergveld in 1970. IEEE Transactions on Biomedical Engineering, 17(1): 70-71 (1970). ISFET is similar to the conventional MOSFET (metal oxide semiconductor field effect transistor), except that the metal layer is replace by an ion-sensitive membrane, an electrolyte solution and a counter electrode. EISFET (electrolyte-insulator-silicon FET) is another acronym that refers to the same structure. The drain source current is modulated by field effect from the ions that can reach the oxide. ISFET technology has been so well-developed that it has made its way to the market as pH meters. Souteyrand et al. is the first to demonstrate label-free-homo-oligomer DNA (18-mer and 1000-mer of poly(dA)DNA) hybridization detection using silicon ISFET. Journal Physical Chemistry B, 101(15): 2980-2985 (1997). They observed a shift in the flat-band potential of the underlying semiconductor in response to the increase of surface charges induced by hybridization between the complementary homo-oligomer strands. Several other papers demonstrating successful field effect DNA detection using silicon ISFET structure are mentioned below. Pouthas et al. demonstrated field effect detection of 5 μM, 10 μM, 20 μM of 20-mer oligonucleotide and emphasized the need for low ionic buffer. Physical Review E, 70(3): 031906 (2004). Fritz et al. were able to detect in real time as dilute as 2 nM of 12-mer oligonucleotide. Proceedings of the National Academy of Science USA, 99(22): 14142-14146. They utilized poly L-lyssine (PLL) to immobilize the probe DNA, and claimed that real time rapid hybridization at low ionic buffer (23 mM phosphate buffer) was enable by the positively charged PLL surface that compensated for electrostatic repulsion between complementary DNA strands. Peckerar et al. demonstrated detection of 1 fM 15-mer DNA. IEEE Circuits & Davies Magazine 19(2): 17-24 (2003).
Thus, current methods for detecting DNA rely upon various combinations of enzymatic amplification of nucleic acids and fluorescent labeling of targets, which entail enzymatic manipulation of the nucleic acid being tested and chemical labeling, respectively. These methods are both time consuming and afford limited sensitivity.
Further, while more recently, DNA microarray technology has been deployed as a tool for monitoring gene expression patterns and profiling of micro RNA (miRNA) in normal and cancerous tissue, quantification of changes has typically been optically-based. While this technique is highly sensitive, use of optical methods impedes progress in both system miniaturization and in direct interfacing with data collection electronics.
Hence, a need exists for a method of detecting DNA that overcomes these disadvantages.